Large novel deletions detected in Chinese families with aniridia: correlation between genotype and phenotype

Purpose To describe the clinical and genetic findings in two Chinese families with aniridia and other ocular abnormalities. Methods Two unrelated families were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Mutation screening of all exons of the PAX6 (paired box gene 6) gene was performed by direct sequencing of PCR-amplified DNA fragments. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions. Linkage analysis was used to validate the large deletions revealed by MLPA in all available family members. Results Clinical examination and pedigree analysis revealed one four-generation family (85) and one three- generation family (86) with total aniridia, congenital cataracts, foveal hypoplasia, and glaucoma. No mutation in PAX6 was identified after PCR-sequencing. Through MLPA analysis, a large deletion including the whole PAX6 gene, DKFZp686k1684 (hypothetical LOC440034), and the RCN1 (reticulocalbin 1) gene was detected in family 85; a 3′ deletion to the PAX6 gene including the ELP4 (elongator complex protein 4) and the DCDC1 (doublecortin domain containing 1) gene was identified in family 86.The two large deletions were confirmed with linkage analysis and the “loss of heterozygous” in the different PAX6 regions were co-segregated with the phenotype of the two families, respectively. Conclusions Patients with the PAX6 contiguous gene deletion, including the RCN1 gene, presented more severe vision impairments than those carrying the PAX6 3′ deletion. Large deletions may account for several Chinese families and sporadic cases with aniridia and screening for these kinds of alterations should be included in aniridia patients’ analyses.

In this study, we describe the clinical findings in two Chinese families with two different large deletions in the region of PAX6.

Patients and DNA sample collection:
This study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects. This study was approved by the Beijing Tongren Hospital Joint Committee on Clinical Investigation, Beijing, China. After informed consents were obtained, participants underwent ophthalmologic examination including bilateral best corrected visual acuity using E decimal charts, slit-lamp biomicroscopy inspection of the anterior chamber, intraocular pressure (IOP) measurement by applanation tonometry (Goldmann), and fundus examination with a 66-diopter VOLK lens. Some patients underwent electroretinography (ERG) and A/B ultrasonic scan examination.
Mutation screening of PAX6: Peripheral blood was obtained by venipuncture and genomic DNA was extracted according to standard protocols. The 14 exons of PAX6 were amplified by polymerase chain reaction (PCR) from genomic DNA. Thirteen pairs of primers for PAX6 were used (Table 1), according to the article previously published [17]. For direct sequencing, PCR products were purified (Shenneng Bocai PCR purification kit; Shenneng, Shanghai, China). An automatic fluorescence DNA sequencer (ABI, Prism 373A; Perkin Elmer, Foster City, CA), used according to the manufacturer's instructions, sequenced the purified PCR products in both forward and reverse directions. DNAssit, version 1.0 compared nucleotide sequences with the published DNA sequence of PAX6 (GenBank NM_001604.3).  Multiplex ligation-dependent probe amplification (MLPA analysis): MLPA was performed with SALSA MLPA Kits P219 (Amsterdam, the Netherlands) according to the manufacturer's instructions. In brief, 100 ng DNA was denatured and hybridized with the SALSA probe mix overnight at 60 °C. The samples with ligase 65 were incubated for 15 min at 54 °C, after which PCR amplification was performed with the specific SALSA FAM PCR primers. The PCR products were separated by capillary electrophoresis on an automatic fluorescence DNA sequencer (ABI, Prism 373A; Perkin Elmer). Data analysis was performed by exporting the peak areas to a Microsoft Excel (Microsoft Corporation, Redmond, WA) file. Each peak was first normalized as described elsewhere [17] and the normalized peak was then divided by the mean of that peak in the control samples. The ratios outside the range of 0.7-1.3 times the control peak area were considered abnormal, with those below 0.7 representing deletions and those above 1.3 representing duplications. For each MLPA analysis, several normal controls were included and the standard deviation for the normal samples was usually less than 10% of the mean. Each result was confirmed by two independent tests.  Table  2. Each assay was done in triplicate. The relative quantitation (RQ) of target gene was accomplished using RQ manager software (Bio Rad systems) and was calculated using the 2 -ddCt method [19]. All experimental samples were normalized using human GAPDH as an internal control. The significance of the difference with a reference experiment was calculated with Student's t-test.

Clinical findings:
We have identified one four-generation family (#85) and one three-generation family (#86) with aniridia. The inheritance pattern in the families was autosomal dominant (Figure 2). After clinical examinations and a review of hospital records, 11 individuals in family 85 were found to have aniridia. All patients presented bilateral complete absence of iris, severe congenital nystagmus, and congenital cataracts ( Figure 3A,C). Foveal hypoplasia was observed in all fourth-generation patients except (IV-6; Figure 3D). The ERG of patient IV-7 showed slight cone cell dysfunction. The proband (III-4), her father, and her brother presented high intraocular pressure (IOP) and late stage glaucoma changes in the optic disc ( Figure 3B). Due to the progressive density of the lens opacification, the fovea of patients in the second and third-generation and patient IV-6 could not be observed clearly. In family 86, five patients were identified and all patients had bilateral complete absence of iris and congenital cataracts ( Figure 3E,G). Neither mental retardation nor other general abnormalities was observed or documented in all patients from the two families. Their detailed clinical features are summarized in Table 3.
Mutation analysis: By the direct sequencing of 14 exons of PAX6, no mutation was detected in the two families. MLPA Results: Using the MLPA Kits P219, two different deletions were detected in the two families ( Figure 4). In family 85, a deletion of the whole PAX6 gene, the DKFZ p686k1684 gene, and the RCN1 gene was found; in family 86, a deletion of the ELP4 gene and the DCDC1 gene, which is located in the 3′ region of the PAX6 gene, was identified.
Genotyping Results: The two families were genotyped with several STRP markers located around PAX6 in the chromosome 11p13 region. The linkage analysis results were highly informative for the two families. For family 85, all patients display "loss of heterozygosis" at marker PAX6 CA/ GT, which is located inside the PAX6 gene. All patients in family 86 show "loss of heterozygosis" at markers D11S995 and D11S2001, which is closed to ELP4 and DCDC1 ( Figure  2). Real-Time Quantitative PCR analysis results: This study set up a real-time quantitative PCR assay to define the relative breakpoint of the deletions. GAPDH (glyceraldehyde-3phosphate dehydrogenase) was used to normalize PAX6 values. Assay for exon 8 of PAX6 were set up by using the patients in family 85 with the PAX6 deletion. The amplification plots are shown in Figure 5A,B, and the patient had about a half RQ value with respect to the normal control Figure 5C. Thus this study used this assay to analyze several single nucleotide polymorphisms (SNPs) around RCN1, PAX6, ELP4, and DCN1. The results were summarized in Table2. The relative exact breakpoints for the two families were showed in Figure 1.

DISCUSSION
In this study, we described two Chinese families with aniridia and other ocular abnormalities. Using MLPA, a large deletion,     including PAX6, DKFZ p686k1684, and RCN1, was identified in family 85; a deletion of ELP4 and DCDC1, leaving PAX6 intact, was found in family 86. The two deletions were cosegregated with the phenotype in the families, respectively. Until now, almost 300 intragenic mutations of PAX6 have been documented in the PAX6 allelic variation database [2][3][4][5][6][7]. Most of the intragenic mutations lead to premature protein truncation, which is likely to be acted on by nonsensemediated decay (NMD). By reviewing the literature on genotype-phenotype correlation studies, the mutations that introduce premature terminated codons (PTCs) are consistently associated with aniridia or closely related phenotypes [6,7]. The patient carrying the complete deletion of PAX6, observed by Vincent et al. [15], did not present distinctive or more severe clinical manifestations than those associated with nonsense mutations. However, more severe bilateral visual impairment was observed in all the patients of family 85. The proband's father and brother totally lost their sight at the age of 40 due to glaucoma. Several patients showed foveal hypoplasia. The large deletion detected in family 85 was novel and contained not only the complete PAX6 gene but also DKFZ p686k1684 and RCN1, which are located about 300 kb upstream of PAX6. RCN1 (reticulocalbin 1), resident in the endoplasmic reticulum, is a Ca 2+ binding protein that participates in the secretory pathway and is expressed in the eye [20].Linkage between Pax6 and Rcn1 has been conserved in mice, humans, and fish [21]. Pax6 was originally isolated in the mouse and mutations in the gene are responsible for the small eye phenotypes. The mouse small eye phenotype had already been suggested through homology mapping to be the mouse counterpart of human aniridia [22]. Recently, Favel et al. [21] observed that the mouse, carrying a heterozygous Pax6 and Rcn1 contiguous deletion, presented an extreme microphthalmia phenotype. They inferred that Rcn1 might directly or indirectly contribute to the eye phenotype in Pax6 contiguous gene deletions. The severe visual impairment observed in family 85 seemed to be consistent with the phenotypes found in the mouse described by Favel et al. [21]. DKFZ p686k1684, located between PAX6 and RCN1, is a non-coding RNA with its function unclear [23].
The 3′ deletion identified in family 86 contained ELP4 and DCD4, which are located downstream of PAX6. The deletions in this region, which contains 3′ regulatory elements for PAX6, were documented in several earlier studies [12][13][14][15][16][17][18]. Most patients harboring the 3′ deletions had only aniridia and other ocular abnormalities, which is similar to the phenotype observed in most nonsense mutations patients. The patients in family 86 showed mild vision impairments due to aniridia and congenital cataracts. Davis et al. [18] described a patient carrying a 1.3 Mb deletion, including several additional genes expressed in the brain, who also presented with autism and mental retardation.
In this study, the aniridia in both families was caused by large deletions in the PAX6 region. In general, patients with PAX6 contiguous deletion, including RCN1, may have relatively severe phenotypes. Large deletions may account for several Chinese families and sporadic cases with aniridia and screening for these kinds of alterations should be included in the aniridia patient's analysis.